Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 22, 2010 (“In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Golpanol BEO
- Physical state: liquid / yellow, clear
- Analytical purity: 99.4%
- pH value: ca. 6 (undiluted test substance)
- Lot/batch No.: 92713124U0
- Expiration date of the lot/batch: 11 Jul 2013
- Stability under test conditions: stable in drinking water over a period of 7 days at ambient temperature.
- Storage condition of test material: Ambient temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes

Test animals

Species:
other: in vitro test on three dimensional human epidermis model (EpiDerm™ model which consists of normal human-derived epidermal keratinocytes cultured to form a multi layered, highly differentiated model of the human epidermis.)
Strain:
other: not applicable
Details on test animals and environmental conditions:
In vitro test system:
- In vitro test system on three dimensional human epidermis models. The EpiDermTM model consists of normal, human-derived epidermal keratinocytes cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
- The test system is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed epidermis after a 1-hour topical exposure and about 42 hours postincubation.

Material and technical equipment:
- EpiDerm™ 200 kit: MatTek Corp., Ashland MA, USA containing 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® diameter 10 mm.

Test system

Type of coverage:
other: not applicable (in vitro test)
Preparation of test site:
other: not applicable (in vitro test)
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro test system: Controls: negative control (NC): 30 μL PBS, sterile; positive control (PC): 30 µL 5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in highly deionized water, sterile
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL bulk volume
Duration of treatment / exposure:
1 hour
Observation period:
42 hours
Number of animals:
not applicable (in vitro test)

Details on study design:
Experimental procedure:
- Direct MTT reduction:
To assess the ability of the test material to directly reduce MTT a pretest was performed. Thereby, the test substance was added to 0.9 mL of the MTT solution. A negative control (highly de-ionized water) was tested concurrently. If the MTT solution colour or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results.

- Basic procedure:
Three tissues were treated with the test substance, the PC and NC, respectively.
• 30 μL of the undiluted test material was applied using a pipette.
• 1 hour after start of application of the test material to the stratum corneum surface of the EpiDermTM tissues, the tissues were washed with sterile PBS to remove residual test material. Subsequently, the tissues were incubated at 37°C for 24 ± 2 hours, transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and thereafter placed into the incubator for additional 18 ± 2 hours post-incubation period.
• After the postincubation period induced tissue destruction (cytotoxicity = loss of viability) was determined by measuring the metabolic activity of the tissue using a colourimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow coloured water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue coloured formazan. Thereby, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.
• After isopropanol extraction of the formazan from the tissues, the optical densitiy of the extract is determined spectrophotometrically. Optical density of the extracts of test substance treated tissues is compared to negative control values and expressed as relative tissue viability.

Acceptance criteria:
In case one of the below given acceptance criteria is not covered, repetition of the test is considered.
- Assay acceptance criterion for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
- Assay acceptance criterion for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Assay acceptance criterion for tissue variability: For every treatment, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

Evaluation criteria:
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
116
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Basis: mean of 3 reconstructed human epidermis model test samples. Time point: 1 hour. Max. score: 125.4. Reversibility: other: reversibility: not applicable. Remarks: In Vitro Skin Irritation . (migrated information)

In vivo

Irritant / corrosive response data:
The test substance is not able to reduce MTT directly.
The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 116%.
Other effects:
No other effects observed.

Any other information on results incl. tables

Table 2: Irritation test

Test

substance

Tissue 1

Tissue 2

Mean

mean

SD

NC

Mean OD570

1.845

1.541

1.747

1.711

 

Viability

(% of NC)

107.8

90.1

102.1

100

9.06

test

item

Mean OD570

2.145

1.840

1.948

1.978

 

Viability

(% of NC)

125.4

107.5

113.9

116

9.04

PC

Mean OD570

0.092

0.098

0.094

0.095

 

Viability

(% of NC)

5.4

5.7

5.5

6

0.19

NC: Negative control (PBS, steril)

PC: Positive control (5% (w/v) sodium dodecyl sulfate (SDS)in highly de-ionized water, sterile

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information
Conclusions:
CLP: not classified
DSD: not classified